Murphy K M, Sweet M J, Hume D A
Centre for Molecular Biology and Biotechnology, University of Queensland, Australia.
J Leukoc Biol. 1994 Sep;56(3):294-303. doi: 10.1002/jlb.56.3.294.
Expression of reporter genes under the control of the HIV-1 long terminal repeat (LTR) was up-regulated in the murine macrophage cell line RAW264 by cotransfection of a plasmid coding for the viral regulatory protein Nef. To determine if a discrete section of the LTR was exclusively responsive to Nef, a series of promoters was produced by successive 5' deletions from the LTR up to the boundary of the enhancer region. These truncated promoters were as active as the full-length sequence in the RAW264 cells, but elimination of the direct repeats and one of the three Sp1 sites reduced promoter activity to minimal levels. Transcription driven by all constructs was equally susceptible to the trans-activating effect of Nef and could be increased further by the addition of a Tat-expressing plasmid to the cotransfection. Open reading frames of nef from NL4-3, from HXB2, which has a premature stop, and a fully functional hybrid of the two under the control of the SR alpha artificial promoter (SV40 early promoter plus HTLV-I R-U5') were able to transactivate the LTR in RAW264 cells to the same degree as HXB3 nef under the control of the cytomegalovirus (CMV) immediate-early promoter. A frameshift mutation of Nef at the XhoI site at position 8475 did not abrogate trans-activation of the LTR in macrophages. To further define the effective trans-activation region of Nef, internal deletions were made. Changes downstream of the XhoI site at amino acid 35 resulted in little or no reduction in trans-activation, whereas a deletion between the CMV promoter of the expression plasmid and the XhoI site largely abolished activity. Nef trans-activation of the LTR may be restricted to macrophages. Parallel cotransfection experiments in COS-1 simian fibroblast-like cells showed repression of reporter expression by Nef. Results suggested that the section of nef responsible for transactivation of the LTR in macrophages differed slightly from that sufficient for trans-repression in fibroblasts. Translation of the protein from the first translation start site (Met-1) rather than from the second in-frame ATG (Met-20) appears to be necessary for the trans-activating effect of Nef in RAW264 cells. Mutation of the initial ATG to ATA led to loss of trans-activating activity. Expression of Nef also has a cytostatic/cytotoxic effect on RAW264 cells indicated by a reduced rate of establishment of stably transfected clones. The cytostatic effect of Nef was not relieved by internal deletions in the coding sequence.(ABSTRACT TRUNCATED AT 400 WORDS)
在鼠巨噬细胞系RAW264中，通过共转染编码病毒调节蛋白Nef的质粒，受HIV-1长末端重复序列（LTR）控制的报告基因的表达上调。为了确定LTR的一个离散区域是否专门对Nef有反应，通过从LTR进行连续的5'缺失直至增强子区域边界，产生了一系列启动子。这些截短的启动子在RAW264细胞中的活性与全长序列相同，但去除直接重复序列和三个Sp1位点之一会将启动子活性降低到最低水平。所有构建体驱动的转录对Nef的反式激活作用同样敏感，并且通过在共转染中添加表达Tat的质粒可进一步增强。来自NL4-3、有一个提前终止密码子的HXB2以及在SRα人工启动子（SV40早期启动子加HTLV-I R-U5'）控制下的两者的完全功能性杂种的nef开放阅读框，能够在RAW264细胞中与在巨细胞病毒（CMV）立即早期启动子控制下的HXB3 nef一样程度地反式激活LTR。Nef在第8475位的XhoI位点处的移码突变并未消除其在巨噬细胞中对LTR的反式激活。为了进一步确定Nef的有效反式激活区域，进行了内部缺失。在氨基酸35处XhoI位点下游的变化导致反式激活几乎没有或没有降低，而表达质粒的CMV启动子与XhoI位点之间的缺失在很大程度上消除了活性。Nef对LTR的反式激活可能仅限于巨噬细胞。在COS-1猿猴成纤维细胞样细胞中的平行共转染实验显示Nef抑制报告基因表达。结果表明，负责在巨噬细胞中反式激活LTR的nef区域与足以在成纤维细胞中进行反式抑制的区域略有不同。从第一个翻译起始位点（Met-1）而非第二个框内ATG（Met-20）翻译蛋白质似乎是Nef在RAW264细胞中产生反式激活作用所必需的。将初始ATG突变为ATA导致反式激活活性丧失。Nef的表达对RAW264细胞也有细胞生长抑制/细胞毒性作用，表现为稳定转染克隆的建立率降低。编码序列中的内部缺失并未缓解Nef的细胞生长抑制作用。（摘要截断于400字）
